CN111118107A - Culture medium for rapid display detection of coliform, preparation method and application - Google Patents
Culture medium for rapid display detection of coliform, preparation method and application Download PDFInfo
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- 239000008101 lactose Substances 0.000 claims abstract description 18
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/10—Enterobacteria
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/245—Escherichia (G)
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Abstract
The invention belongs to the technical field of microbial culture, and discloses a culture medium for rapid display detection of coliform groups, a preparation method and application thereof, wherein the culture medium for rapid display detection of the coliform groups consists of 5-10 g of yeast powder, 5-15 g of tryptone, 5-15 g of lactose, 5-10 g of sodium chloride, 0.01-0.04 g of bromocresol purple and 0.0001-0.1 g of thiazole blue, and water is used for supplementing to 1000 ml. The coliform group bacteria detection kit and the detection paper sheet both comprise a culture medium for quickly culturing and developing the coliform group bacteria. The detection method for rapid display detection of coliform bacteria is realized by applying a coliform bacteria detection kit and a detection paper sheet. The detection method for rapid display detection of coliform bacteria provided by the invention has the advantages of high selectivity, short detection period, low cost, strong operability, simple operation, less time consumption and high sensitivity, and is suitable for rapidly, simply and efficiently separating and identifying the coliform bacteria.
Description
Technical Field
The invention belongs to the technical field of microbial culture, and particularly relates to a culture medium for rapid display detection of coliform, a preparation method and application.
Background
Currently, the closest prior art: the coliform group, which is not a designation for bacteriological classification but a term in the field of hygienic bacteria, does not represent a certain bacterium or a certain genus of bacteria, but refers to a group of bacteria associated with fecal contamination having certain characteristics, which are not completely identical in terms of biochemistry and serology, and are defined as: aerobic and facultative anaerobic gram-negative bacillus capable of decomposing lactose to produce acid and gas at 37 deg.c. It is generally recognized that the bacteria of this group may include Escherichia coli, Citrobacter, Klebsiella aerogenes, Enterobacter cloacae, and the like.
The coliform group mainly includes the genera Escherichia, Citrobacter, Klebsiella and Enterobacter in the family Enterobacteriaceae. Bacteria of these genera are both from the intestinal tract of humans and warm-blooded animals, aerobic and facultative anaerobic, do not form spores; can ferment lactose, lactic acid, lactic. The coliform group is mainly the genus Escherichia coli, which is commonly called as classical Escherichia coli. Coliform bacteria are faeces from humans and warm-blooded animals, either directly or indirectly. The genera in the group except typical Escherichia coli can be directly derived from feces, and can also be derived from the variation of the typical Escherichia coli in the environment 7-30 days after being discharged from the body. The detection of coliform groups in food products therefore indicates that the food product is contaminated with faeces from humans or warm-blooded animals, where typical coliform bacteria are recent contamination of faeces and other bacteria may be old contamination of faeces.
There are many methods for detecting coliform bacteria, and conventional bacteriological culture methods, immunological methods, nucleic acid probe techniques, and the like are mainly relied on. The bacteriological culture method needs to be subjected to enrichment culture, selective separation, morphological characteristic observation, physiological and biochemical reaction, serological identification and other processes, generally needs 4 to 7 days, is complex to operate, time-consuming and labor-consuming, has low sensitivity and poor specificity, and cannot detect damaged bacteria and dead bacteria; the immunological method is easy to pollute, and has more serious cross reaction, more false positive and lower sensitivity; the biggest advantage of the nucleic acid probe detection technology is strong specificity, but the probe detection technology has certain problems, such as low sensitivity; a probe is prepared for detecting one bacterium.
The paper sheet method is also a commonly used method for rapidly detecting coliform groups. The paper sheet method is based on the principle that lactose is decomposed to produce acid when coliform bacteria grow and develop, dehydrogenase is generated to dehydrogenate, and hydrogen and colorless triphenyltetrazolium chloride (TTC) react to form an insoluble red compound (triphenylmethylhydrazone, TTF) to enable bacterial colonies (lawn) to turn red, and a certain amount of lactose, indicators (TTC, acid-base indicators), peptone and the like are adsorbed on sterile filter paper with a specific area. The coliform group showed the intrinsic characteristics of acid production by fermented lactose, yellowing of the paper and formation of red spots (halos) by the 2 indicators mentioned above. This property was used as the sole criterion for positive results. However, in the detection of food, seasoning and other samples containing pepper, the red pepper particles contained therein are very similar to red coliform colonies, so that false positive discrimination occurs and the counting result is seriously disturbed.
Therefore, there is a need in the art for a method for detecting coliform bacteria that is efficient, rapid, inexpensive, accurate, and easy to operate.
In summary, the problems of the prior art are as follows:
(1) the bacteriological culture method needs to be subjected to enrichment culture, selective separation, morphological characteristic observation, physiological and biochemical reaction, serological identification and other processes, generally needs 4 to 7 days, is complex to operate, time-consuming and labor-consuming, has low sensitivity and poor specificity, and cannot detect damaged bacteria and dead bacteria.
(2) The immunological method is easy to pollute, and has more serious cross reaction, more false positive and lower sensitivity.
(3) The sensitivity of the nucleic acid probe detection technology is not high enough; a probe is prepared for detecting one bacterium.
(4) The paper method can realize the rapid detection of coliform group bacteria, but cannot eliminate the influence of red pepper particles in samples containing pepper food, seasonings and the like on result judgment.
Therefore, there is a need in the art for a rapid, accurate, and easy-to-operate method for detecting coliform bacteria, particularly for detecting coliform bacteria that can be applied to samples containing capsicum foods, condiments, and the like and can eliminate interference of red capsicum particles.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a culture medium for rapid display detection of coliform bacteria, a preparation method and application thereof.
The culture medium for the rapid display detection of the coliform group is prepared from 5-10 g of yeast powder, 5-15 g of tryptone, 5-15 g of lactose, 5-10 g of sodium chloride, 0.01-0.04 g of bromocresol purple and 0.0001-0.1 g of thiazole blue, and water is used for supplementing to 1000 ml.
Note: thiazole Blue (MTT) is known as 3- (4,5-dimethyl-2-Thiazolyl) -2,5-diphenyl-2-H-tetrazolium bromide, Thiazolyl Blue tetrazolium bromide, chinese chemical name is 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide, trade name: thiazole blue, a yellow dye.
The invention also aims to provide a preparation method of the culture medium for rapid displaying and detecting of the coliform group, which comprises the following steps:
(1) preparing a liquid culture medium: weighing yeast powder, tryptone, lactose, sodium chloride, bromocresol purple and thiazole blue according to the formula respectively, fixing the volume to 1000mL, preparing a culture medium, and sterilizing for later use.
(2) Preparing a solid culture medium: weighing yeast powder, tryptone, lactose, sodium chloride, bromocresol purple, thiazole blue and agar according to the formula respectively, diluting to 1000mL, preparing a culture medium, sterilizing, and pouring into a flat plate for later use.
The invention also aims to provide a coliform group detection kit applying the culture medium for the quick displaying detection of the coliform group, and the coliform group detection kit comprises the culture medium for the quick culturing and color development of the coliform group.
The invention also aims to provide a coliform group detection paper sheet applying the culture medium for the quick display detection of the coliform group, and the coliform group detection paper sheet comprises the culture medium for the quick culture and color development of the coliform group.
The invention also aims to provide a detection method for rapid display detection of coliform group by applying the culture medium for rapid display detection of coliform group.
The invention also aims to provide a detection method for rapid display detection of coliform group by applying the coliform group detection paper of the coliform group detection kit.
The invention also aims to provide a detection method for rapid display detection of coliform group by applying the coliform group detection paper.
The invention also aims to provide an application of the culture medium for rapid display detection of coliform group in preparation of a diagnostic agent for detecting coliform group.
The invention also aims to provide application of the coliform group detection kit in preparation of a diagnostic agent for detecting coliform groups.
The invention also aims to provide an application of the coliform group detection paper sheet in preparation of a diagnostic agent for detecting coliform groups.
The invention also aims to provide the application of the detection method for the rapid display detection of the coliform group in the aspects of medical microorganism samples, medicines, medical instrument health inspection samples, public health monitoring samples, foods, seasonings, cosmetics and industrial product detection.
The invention also aims to provide the application of the diagnostic agent for detecting coliform bacteria in the aspects of medical microorganism samples, medicines, medical instrument health inspection samples, public health monitoring samples, foods, seasonings, cosmetics and industrial product detection.
In summary, the advantages and positive effects of the invention are: the detection method for rapid display detection of coliform bacteria provided by the invention has the advantages of high selectivity, short detection period, low cost, strong operability, simple operation, less time consumption and high sensitivity, and is suitable for rapidly, simply and efficiently separating and identifying the coliform bacteria. The culture medium for rapid display detection of coliform groups provided by the invention has better specificity, the coliform groups have higher growth speed on the culture medium, and rapid identification can be carried out through color, so that the specific identification of the coliform groups can be realized respectively.
Drawings
FIG. 1 is a flow chart of a preparation method of a culture medium for rapid display detection of coliform bacteria according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a detection method for rapid display detection of coliform group, and the invention is described in detail with reference to the attached drawings.
The culture medium for rapid display detection of coliform groups provided by the embodiment of the invention comprises 5-10 g of yeast powder, 5-15 g of tryptone, 5-15 g of lactose, 5-10 g of sodium chloride, 0.01-0.04 g of bromocresol purple and 0.0001-0.1 g of thiazole blue, and water is used for supplementing to 1000 ml.
As shown in fig. 1, a method for preparing a culture medium for rapid display detection of coliform bacteria according to an embodiment of the present invention includes:
(1) preparing a liquid culture medium: weighing yeast powder, tryptone, lactose, sodium chloride, bromocresol purple and thiazole blue according to the formula respectively, fixing the volume to 1000mL, preparing a culture medium, and sterilizing for later use.
(2) Preparing a solid culture medium: weighing yeast powder, tryptone, lactose, sodium chloride, bromocresol purple, thiazole blue and agar according to the formula respectively, diluting to 1000mL, preparing a culture medium, sterilizing, and pouring into a flat plate for later use.
The embodiment of the invention provides a coliform group detection kit which comprises a culture medium for quickly culturing and developing the coliform group.
The paper sheet for detecting the intestinal flora provided by the embodiment of the invention comprises a culture medium for quickly culturing and developing the intestinal flora.
The detection method for rapid display detection of coliform bacteria provided by the embodiment of the invention is realized by applying the culture medium for rapid display detection of coliform bacteria.
The detection method for rapid display detection of coliform bacteria provided by the embodiment of the invention is realized by applying a coliform bacteria detection paper sheet of a coliform bacteria detection kit.
The detection method for rapid display detection of coliform bacteria provided by the embodiment of the invention is realized by applying a coliform bacteria detection kit.
The culture medium for rapid display detection of coliform bacteria provided by the embodiment of the invention can be applied to preparation of a diagnostic agent for detecting the coliform bacteria.
The coliform group detection kit provided by the embodiment of the invention can be applied to the preparation of a diagnostic agent for detecting coliform groups.
The coliform group detection paper provided by the embodiment of the invention can be applied to the preparation of a diagnostic agent for detecting coliform groups.
The detection method for rapid display detection of coliform bacteria provided by the embodiment of the invention can be applied to detection of medical microorganism samples, medicines, medical instrument health inspection samples, public health monitoring samples, food, seasonings, cosmetics and industrial products.
The diagnostic agent for detecting coliform bacteria provided by the embodiment of the invention can be applied to detection of medical microorganism samples, medicines, medical instrument health inspection samples, public health monitoring samples, foods, seasonings, cosmetics and industrial products.
The detection method for rapid display detection of coliform bacteria provided by the embodiment of the invention can be applied to detection of medicines, foods, cosmetics and industrial products.
The technical solution of the present invention is further described with reference to the following examples.
Example 1: preparation of culture medium
Preparing a liquid culture medium: weighing the components according to the formula of 6g of yeast powder, 12g of tryptone, 8g of lactose, 9g of sodium chloride, 0.01g of bromocresol purple and 0.003g of thiazole blue, diluting to 1000mL, preparing a culture medium, and sterilizing for later use.
Preparing a solid culture medium: weighing the components according to the formula of 6g of yeast powder, 12g of tryptone, 8g of lactose, 9g of sodium chloride, 0.01g of bromocresol purple, 0.003g of thiazole blue and 18g of agar, diluting to 1000mL, preparing a culture medium, sterilizing, and pouring into a flat plate for later use.
Example 2: comparative test of coliform group chromogenic medium and paper slip method in prior art for testing Pi county broad bean paste
1. The chromogenic medium plate of the invention is prepared by: prepared according to the method of example 1.
2. Control was performed using 3MTMPetrifilmTM6412 quick test piece for coliform bacteria.
3. Sample processing
Weighing 25g of Pi county bean cotyledon sample, putting the Pi county bean cotyledon sample into an aseptic homogenizing cup containing 225mL of phosphate buffer solution or normal saline, homogenizing for 1min to 2min at 8000r/min to 10000r/min, or putting the Pi county bean cotyledon sample into an aseptic homogenizing bag containing 225mL of phosphate buffer solution or normal saline, beating for 1min to 2min by using a beating type homogenizer, and preparing into a product 1: sample aliquot of 10.
The pH of the sample homogenizing solution is 6.5-7.5, and is adjusted by 1mol/L NaOH or 1mol/L HCl respectively when necessary.
Pipette 1: 10 samples are homogenized for 1mL, 9mL phosphate buffer solution or physiological saline sterile test tubes are slowly injected into the tube wall (the tip of a suction tube or a suction head does not touch the dilution liquid level), the test tubes are shaken or repeatedly blown and beaten by using 1 piece of 1mL sterile suction tube to mix uniformly, and 1: 100 sample aliquots.
According to the estimation of the sample pollution condition, ten times of serial diluted sample homogenizing solutions are sequentially prepared according to the operation. Each incremental dilution was 1 time, replaced with 1mL sterile pipette or pipette tip. From the preparation of the sample homogenizing liquid to the completion of the sample inoculation, the whole process cannot exceed 15 min.
4. Inoculating and culturing
2 to 3 suitable serial dilutions were selected, each dilution being inoculated into 2 sterile plates containing the medium of the invention and control paper sheets, 1mL per plate. Meanwhile, 1mL of physiological saline was used as a blank control. Culturing at 36 +/-1 deg.c for 18-24 hr.
5. Analysis of results
The results of the experiment are shown in table 1.
TABLE 1 results of the experiment
It can be seen that the culture medium of the present invention has the ability to specifically identify coliform group, which has a fast growth rate on the culture medium and can be identified rapidly by color.
Example 3: test on the inspection accuracy of the color development culture medium of coliform group and the paper slip method in the prior art for the Pixian bean paste
The single colonies cultured in example 2 were picked up and inoculated into Brilliant Green lactose bile salt (BGLB) broth tubes, cultured at 36 + -1 deg.C for 24-48 h, and gas production was observed. The BGLB broth tube produces gas, and the result is reported to be positive coliform.
The identification results are as follows:
the culture medium of the invention: 50 blue-violet colonies are picked, 50 results are positive, and the accuracy rate is 100%;
commercially available rapid test paper: 50 red colonies are picked, 45 are positive, and the accuracy rate is 90%;
therefore, the culture medium has better specificity, and can realize specific identification of coliform groups, particularly coliform groups in seasonings.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. The culture medium for the rapid display detection of the coliform group is characterized by comprising 5-10 g of yeast powder, 5-15 g of tryptone, 5-15 g of lactose, 5-10 g of sodium chloride, 0.01-0.04 g of bromocresol purple and 0.0001-0.1 g of thiazole blue, and water is used for supplementing to 1000 ml.
2. The method for preparing the culture medium for rapid display detection of coliform bacteria according to claim 1, wherein the culture medium for rapid display detection of coliform bacteria is prepared by the following steps:
(1) preparing a liquid culture medium: weighing yeast powder, tryptone, lactose, sodium chloride, bromocresol purple and thiazole blue according to the formula respectively, fixing the volume to 1000mL, preparing a culture medium, and sterilizing for later use;
(2) preparing a solid culture medium: weighing yeast powder, tryptone, lactose, sodium chloride, bromocresol purple, thiazole blue and agar according to the formula respectively, diluting to 1000mL, preparing a culture medium, sterilizing, and pouring into a flat plate for later use.
3. Use of the medium for rapid display of coliform bacteria according to claim 1 in a rapid display of coliform bacteria.
4. Use of the medium for rapid demonstration of coliform group bacteria according to claim 1 in the preparation of a diagnostic for detecting coliform group bacteria.
5. A coliform group detection kit comprising the medium for rapid display of coliform group detection according to claim 1.
6. Use of the coliform group detection kit of claim 5 in a rapid demonstration test of coliform groups.
7. Use of the coliform group detection kit of claim 5 in the preparation of a diagnostic for detecting coliform groups.
8. A strip of coliform bacteria comprising the medium for rapid demonstration of coliform bacteria according to claim 1.
9. Use of the coliform bacteria detection paper of claim 7 in a rapid coliform bacteria display test.
10. Use of the coliform group detection paper of claim 6 in the preparation of a diagnostic agent for detecting coliform groups.
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Citations (5)
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|---|---|---|---|---|
| JPH0451900A (en) * | 1990-06-15 | 1992-02-20 | Nitsusui Seiyaku Kk | Culture medium for detecting coliform group |
| CN1718741A (en) * | 2005-06-23 | 2006-01-11 | 王彩红 | Method of fast detecting colibacillus in beer using paper |
| CN101013127A (en) * | 2007-01-05 | 2007-08-08 | 山东省疾病预防控制中心 | Foodstuff coliform group bacteria rapid detecting paper |
| CN101200755A (en) * | 2007-12-05 | 2008-06-18 | 华南农业大学 | Bacterium identification reagent kit as well as preparation method and uses thereof |
| CN102888442A (en) * | 2011-07-20 | 2013-01-23 | 农高惠 | Kit for rapid culture, identification and drug sensitivity of gastric helicobacter pylori and inspection method thereof |
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| JPH0451900A (en) * | 1990-06-15 | 1992-02-20 | Nitsusui Seiyaku Kk | Culture medium for detecting coliform group |
| CN1718741A (en) * | 2005-06-23 | 2006-01-11 | 王彩红 | Method of fast detecting colibacillus in beer using paper |
| CN101013127A (en) * | 2007-01-05 | 2007-08-08 | 山东省疾病预防控制中心 | Foodstuff coliform group bacteria rapid detecting paper |
| CN101200755A (en) * | 2007-12-05 | 2008-06-18 | 华南农业大学 | Bacterium identification reagent kit as well as preparation method and uses thereof |
| CN102888442A (en) * | 2011-07-20 | 2013-01-23 | 农高惠 | Kit for rapid culture, identification and drug sensitivity of gastric helicobacter pylori and inspection method thereof |
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